Disposable cartridge for characterizing particles suspended in a liquid

ABSTRACT

A particle characterization apparatus in which particles suspended in a liquid pass through an orifice or aperture for detection and characterization of the particles utilizing impedance determination. In particular, the apparatus includes a membrane of a polymer as a base material for precision machining of a sub-millimeter orifice. Forming the orifice in a polymer membrane facilitates the construction of a single use cartridge for haematology analysis due to low material and production costs.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. 119 to Danishapplication PA 2003 00159 filed on Feb. 5, 2003, and under 35 U.S.C.119(e)(1) to U.S. Provisional application Ser. No. 60/387,407 filed Jun.11, 2002, which are hereby incorporated by reference.

The present invention relates to a particle characterization apparatusin which particles suspended in a liquid passes through an orifice oraperture for detection and characterization of the particles utilisingimpedance determination. In particular the invention relates toutilisation of a membrane of a polymer as a base material for precisionmachining of a sub-millimetre orifice. Forming the orifice in a polymermembrane facilitates the construction of a single use cartridge forhaematology analysis due to low material and production costs.

Automated Blood Analysers (Haematology Analysers) are based onelectrical or optical ways of characterizing each individual blood cellon the fly in a fluid flow. Such instrumentation is rather sophisticatedand requires trained personnel to perform the measurements. Counting andsizing of particles by impedance cell sizing, also known as CoulterSizing or Coulter counting (see V. Kachel, “Electrical Resistance PulseSizing: Coulter Sizing”, Flow Cytometry and Sorting, Second Edition, pp.45-80, 1990 Wiley-Liss), is a broadly accepted method that is being usedin most haematology-analysers and particle counting equipment. Themethod is based on measurable changes in the electrical impedanceproduced by comparatively non-conductive particles in an electrolyte. Asmall opening called the “aperture” or “orifice” connects twoelectrically isolated chambers, each having electrodes for contactingthe electrolyte. The orifice works as a restriction to the electricalpath, whereby a sensing zone is established through which the particlesare aspirated. In the sensing zone each particle will give rise to adisplacement of the surrounding electrolyte, thus blocking part of thecurrent-path and giving rise to a short voltage pulse. By aspiration ofparticles one by one through the orifice, the particles can becharacterized with respect to volume and conductivity by registration ofthe pulse characteristics. The concentration of specific subgroups ofthe particles may be determined from the pulse characteristics and bymetering the analysed sample volume.

Conventional instruments utilizing the impedance technique are based ona fixed membrane with a precision-machined orifice, which is beingmaintained by flushing and rinsing the membrane. In conventionalimpedance cell-sizing equipment, the orifice is made in a membrane ofsapphire by micro drilling and polishing. These orifices show excellentrobustness and may be used repeatedly for several thousand analyses whencleaned properly in between. However, being made of sapphire andrequiring cumbersome techniques of preparation, these membranes arefairly expensive to replace.

WO 01/69292 discloses a portable haematology analyser with amaintenance-free reader and a unique disposable cartridge for bloodsampling and handling. The disposable cartridge includes a membrane withan orifice for impedance cell sizing.

In order to be able to provide single-use cartridges, there is a needfor a cheap, single-use membrane material with a precision-machinedorifice.

Further, there is a need for a method of producing a membrane with anorifice with accuracy and reproducibility at low cost

Typically, the known methods do not provide the required accuracy. Inorder to facilitate accurate impedance determination, it is desired thatthe accuracy of the diameter of the orifice lie within +/−10%, morepreferably within +/−5% and more preferably within +/−2%. The desireddiameter of the orifice is typically in the range from 10 μm to 1000 μm,preferably in the range from 30 μm to 75 μm. Thus, the manufacturingprocess must therefore be able to provide orifices with a precision inthe μm-scale, e.g. within +/−2 μm accuracy in order for the manufacturedmembrane with orifice to provide useful results.

Thus, it is an object of the present invention to provide a membranewith an orifice for use in an Impedance cell sizing apparatus, e.g. witha single-use cartridge, for characterizing particles suspended in aliquid, e.g. cells in a blood sample. Preferably, the cartridge enablessample taking, sample preparation, and particle characterization so thatanalysis may be performed within one device without a need for samplehandling and sample transfer to another unit.

The single-use cartridge is intended to be discarded after analysis ofone liquid sample.

According to the present invention, the above-mentioned and otherobjects are fulfilled by a method of producing an orifice in a polymermembrane by precision machining, such as milling, drilling, punching,ablation, evaporation, injection moulding, punching, water cutting, aircutting, laser cutting, etc.

The use of a polymer sheet has proved to be the ideal way of meeting therequest for a disposable cartridge for cell analysis. The majoradvantages of the polymer sheet are the low cost of the material, thelow cost of the manufacturing process, simple and reliable weldingmethods with other polymers, ideal electrical insulation characteristicsand good chemical stability.

Soft polymers, such as photopolymers (photoresist), are soft or fluid innature and must be applied on a supporting surface before hardening. Themembrane thickness of such photopolymers is difficult to control and mayvary significantly over the entire surface on which it has been applied.In order to yield control of the thickness over a larger area of themembrane, the membrane must be fabricated by using rolls to define therequired thickness.

Thus, preferably, the polymer membrane is manufactured from a hardenedpolymer or a hard polymer so that the membrane is self-sustained.

Photolithography is a slow process with many different process stepssuch as pre-heating, exposure, curing and dissolving, which makes thisfabrication method cumbersome and expensive.

Thus, preferably, the precision machining of the membrane comprisesother processes than photolithography.

Manufacturing of polymer membranes with orifices by laser cuttingaccording to the present invention provides cheap and rapid productionof membranes with precision-machined orifices for impedance particlecounting and/or sizing.

The high-energy laser spot causes vaporization or ablation of thematerial in the focused region of the spot. The laser spot of an excimerlaser may be focused to a few micrometers, providing the desiredaccuracy in accordance with the present invention.

Preferably, the laser is a UV-laser because of its superior lasercutting accuracy.

Preferably, the UV-laser is an excimer laser with a wavelength in therange from 150 nm to 350 nm.

According to one embodiment of the invention, the laser is used like aconventional drill, i.e. the focussed laser spot remains at the desiredposition of the orifice and the orifice are produced by a series oflaser pulses.

According to another embodiment of the invention, the focussed laserbeam is scanned along the desired circumference of the orifice therebycutting-out the orifice of the membrane. In this way any desiredcircumferential shape of the orifice may be manufactured.

According to yet another embodiment of the invention, a narrow laserbeam is scanned for example linearly, e.g. line by line, across thesurface of the membrane desired to be removed for creation of theorifice.

According to a second aspect of the invention, a polymer membrane isprovided with an orifice with rounded edges at one of the sides of themembrane whereby perturbations of an electrical field at the orificeentrance are minimised and a substantially homogenous electrical fieldat the centre of the orifice may be provided.

Hereby, electrical pulses generated by particles passing the orifice atthe centre of the orifice and particles passing the orifice close to anedge of the orifice will generate substantially identical pulses.Without rounded edges, particles passing the orifice close to an edgewill generate a distorted pulse.

Preferably, the radius of curvature of the rounded edges corresponds to¼th of the diameter of the orifice with a length to diameter ratio of 1.Hereby, a homogeneous field is still reached in the orifice with nofield distortion at the edge.

In order to establish the rounded edges of the orifice, the laser isprogrammed to process a larger area in the beginning and then narroweddown to the diameter defining the effective diameter of the orifice.

Further, a polymer membrane is provided with an orifice with a surfaceroughness of its internal surface in the range from 0 μm to 5 μm wherebya substantially homogenous electrical field at the centre of the orificemay be provided.

Still further, a polymer membrane is provided with an orifice with adeviation of the orifice diameter along a longitudinal axis of theorifice in the range from +/−1% to +/−10% whereby a substantiallyhomogenous electrical field at the centre of the orifice may beprovided.

The membrane according to the present invention may for example beincorporated into a cartridge for characterizing particles suspended ina liquid, comprising a housing with a mixing chamber and a collectionchamber separated by the membrane containing the orifice for passage ofthe particles between the mixing chamber and the collection chamber.Particle characterization means are provided for characterizingparticles passing through the orifice.

Sample taking may be performed through a bore in the outer surface ofthe housing for entrance of a liquid sample. The housing furthercomprises a sampling member that is movably positioned in the housing.The sampling member has a cavity for receiving and holding a small andprecise volume of liquid. In a first position of the sampling member,the cavity is in communication with the bore for entrance of the liquidsample into the cavity, and, in a second position of the samplingmember, the cavity is in communication with an inlet to the mixingchamber.

Thus, the sampling member operates to receive and hold a precise volumeof liquid sample and to transfer the sample to the inlet of the mixingchamber.

Preferably, liquid to be sampled enters the cavities by capillaryattraction causing a liquid flow. Utilization of capillary forcessimplify the flow system, since no pumps, membranes, syringes or otherflow generating means are needed to take the sample.

Thus, the bore may form a first capillary tunnel for entrance of aliquid sample by capillary attraction. The capillary tunnel isdimensioned so that, upon contact between the bore and liquid to besampled, a sample of the liquid is drawn into the bore by capillaryattraction.

Further, the cavity may form a second capillary tunnel adapted fordrawing the liquid sample into the cavity by capillary attraction.Preferably, the first and second capillary tunnel has the same diameter,and it is also preferred that, in the first position, the first andsecond capillary tunnel extend along substantially the same longitudinalcentre axis.

Preferably, the sampling member is rotatable about an axis of rotationthat is substantially perpendicular to a longitudinal axis of thecavity.

Additionally or alternatively, the sampling member may be displaced in adirection substantially perpendicular to a longitudinal axis of thecavity.

The surface of the first and second inner capillary tunnel walls may behydrophilic whereby the capillary attraction of the liquid sample isfacilitated. For example, the inner tunnel walls may be made of e.g.glass or polymers, such as polystyrene.

Alternatively, the capillary tunnel walls may be made of another type ofmaterial and covalently or non-covalently coated with a hydrophilicmaterial, such as a polymer or a reagent.

The capillary tunnel may also include one or more reagents adhered orchemically bonded to the inner tunnel wall. These reagents serve thepurposes of further facilitating the capillary attraction of the sampleand optionally also causing a chemical reaction in the liquid sample,e.g. introducing anticoagulant activity in a blood sample. Such reagentsmay comprise heparin, salts of EDTA, etc.

Preferably, the sampling member is made of a polymer.

In accordance with a further aspect of the invention, an apparatus isprovided for characterizing particles suspended in a liquid, comprisinga cartridge as disclosed herein, and a docking station for removablyreceiving the cartridge, the docking station comprising connectors foroperational connection with the particle characterization means when thecartridge is received in the docking station.

The cartridge may further comprise a cartridge port communicating withthe collection chamber for causing a liquid flow through the orifice,and the docking station may further comprise a corresponding port forforming a gas connection with the cartridge port when the cartridge isreceived in the docking station for application of a pressure causing aliquid flow through the orifice.

The particle characterization means may include a first electrode in themixing chamber and a second electrode in the collection chamber, eachelectrode being electrically connected to a respective terminal memberaccessible at the outer surface of the cartridge for operationalconnection to the respective connector of the docking station when thecartridge is received in the docking station. Generally, it is preferredthat all necessary electrical and fluid connections to the cartridge canbe established by fitting the cartridge into the docking station,preferably by a simple push fit.

The first and second electrodes may facilitate particle characterizationutilizing the well-known Coulter impedance principle, e.g. for countingand sizing of blood cells. This method has become a globally acceptedmethod and is being used in the majority of haematology-analysers.Several thousand particles per second may be characterized with highprecision and accuracy utilizing this principle.

With the electrical impedance technique it is possible to resolve theparticle volume from the measurement. By maintaining a constant currentacross the orifice, the recorded voltage pulse from particles displacingthe electrolyte in the orifice will have a height proportional to thevolume of the particle provided that the particles can be considerednon-conducting compared to the electrolyte, the electrical field (DC orRF) in the centre of the orifice can be considered homogeneous, whichtypically is fulfilled when the diameter D is smaller than the length lof the orifice (l/D>1), the particle d can be considered small comparedto the diameter of the orifice (d<0.2*D), and that only one particlepasses through at a time and the particles are passed through theorifice in along the length of the orifice.

Preferably, the length or depth of the orifice is from 1 to 1000 μm, forexample about 50 μm. Desirably the length of the orifice is chosen suchthat only one particle will be present in the orifice at the time whendetecting particles of from 0.1 to 100 μm diameter. However,considerations to the homogeneity of the electrical field in the orificemay require a length of the orifice larger or equal to the diameter. Thecounts, of which some may be simultaneous counting of two particles, canbe corrected mathematically by implementing a statistical estimation.The aspect ratio of the orifice, (length or depth divided by diameter)is preferably from 0.5:1 to 5:1, more preferably from 1:1 to 3:1.

Preferably, the largest cross-sectional dimension of the orifice is from5 to 200 μm, for example 10 to 50 μm.

The cartridge may further comprise a breather inlet/outlet communicatingwith the surroundings for preservation of substantially ambientatmospheric pressure in the cartridge flow system for facilitation ofliquid flow through the orifice.

Preferably, the cartridge is designed to be disposable after a singleuse. It is desirable that after use there is no need to clean theapparatus before it can be used in a new assay procedure with a newcartridge. Accordingly, escape of liquid from the cartridge at its entryinto the docking station should be avoided. To this end the positioningof the orifice with respect to the breather inlet/outlet, the secondchamber inlet/outlet and the particle characterization device componentsis preferably such that a volume of liquid sufficient for the desiredparticle characterization can be drawn or pumped through the orificewithout the liquid passing out of the housing. Generally, it should bepossible to pass a volume of liquid, which is at least 0.1 ml to 10 ml,e.g. 0.5 ml, through the orifice whilst particle characterizationmeasurements are being made with no liquid leaving the cartridge.

The cartridge may comprise volume-metering means for determining thebeginning and end of a period during which a predetermined volume ofliquid has passed through the orifice.

Preferably, the volume metering means comprises a volume-meteringchamber with an input communicating with the collection chamber and anoutput, and wherein presence of liquid is detected at the input and atthe output, respectively.

For example, presence of liquid may be detected optically due to changedoptical properties of a channel configuration from being filled with airtill when it is being filled with liquid. This could be constructed asreflectance or transmittance detection from the surface, where incidentlight is reflected from an empty channel and transmitted through afilled channel, thus giving a clear shift in the detected reflected ortransmitted light.

It is preferred that the input and output of the metering chamber isformed by narrow channels for accommodation of only a small liquidvolume compared to the volume of the metering chamber so that the actualpositioning of the volume metering means, e.g. optical reflectancedetection, in the channels do not substantially influence the accuracyof the volume metering means determination.

The mixing chamber or the collection chamber may constitute the volumemetering chamber; however, it is preferred to provide an independentvolume metering chamber facilitating positioning of the volume meteringmeans, e.g. the optical reflectance detection.

The volume metering means may be positioned for sensing when liquid inthe metering chamber is at or above respective levels in thevolume-metering chamber.

The volume metering means may be used for sensing when the level of theliquid is such that the respective metering means are or are not filledwith the liquid and may therefore serve for determining the beginningand end of a period during which a fixed volume of liquid has passedthrough the orifice. For example, particle characterization may beginwhen the level of the liquid just rises over the level of a meteringmeans and may end when the level of the liquid just rises over a secondmetering means, the volume of liquid passing through the orifice duringthis period being defined by the separation of the respective meteringmeans.

Where the end point of the passage of a defined volume of liquid is theeffective emptying of one chamber to below the level of the orifice, itis preferred that each of the collection and mixing chambers (or atleast that chamber from which liquid passes) has a transverse crosssectional area at the level of the orifice which is substantially lessthan the transverse cross sectional area of the chamber over asubstantial part of the height of the chamber above the orifice.

When using the Coulter principle the diluent for use in the apparatusaccording to the invention may contain inorganic salts rendering theliquid a high electrical conductivity. When sample is applied to theelectrolyte, the electrolyte to sample volumes should preferably behigher than 10. Sample preparation should preferably result in between1,000 to 10,000,000 particles per ml and more preferably between 10,000and 100,000 particles per ml. A mixing of the sample after addingelectrolyte is recommended. Particle diameters should preferably bewithin 1 to 60 percent of the orifice diameter and more preferablybetween 5 to 25 percent of the orifice diameter. Volume flow shouldpreferably be from 10 μl to 10 ml per minute and more preferably between100 μl and 1 ml per minute. For the measurement a constant electricalcurrent of approximately 1 to 5 mA should preferably be applied. Thesource of electrical current should preferably have a signal to noiseratio (S/N) better than 1,000. The response from the electrode can befiltered electronically by a band-pass filter.

The invention will be further described and illustrated with referenceto the accompanying drawings in which:

FIG. 1 is a cross sectional side view through the components of adisposable cartridge according to the present invention,

FIG. 2 schematically illustrates the flow-through sensor concept,

FIG. 3 schematically illustrates an apparatus according to the presentinvention with the disposable cartridge, a docking station, and areader,

FIG. 4 is a plot of results obtained in Example 1,

FIG. 5 is a plot of results obtained in Example 2,

FIG. 6 is a plot of results obtained in Example 3,

FIG. 7 is a plot of results obtained in Example 4,

FIG. 8 is a plot of results obtained in Example 5,

FIG. 9 schematically illustrates manufacturing of a membrane with anorifice according to an embodiment of the present invention,

FIG. 10 schematically illustrates manufacturing of a membrane with anorifice according to another embodiment of the present invention, and

FIG. 11 shows a cross-section of a membrane orifice manufactured inaccordance with the present invention.

FIG. 1 shows a disposable cartridge with a housing 85 for bloodanalysis, comprising a liquid storage chamber 1 containing a liquiddiluent 11, a sampling member 2 positioned in the housing 85 forsampling a blood sample 8 and having a cavity 10 for receiving andholding the blood sample 8, the member 2 being movably positioned inrelation to the housing 85 in such a way that, in a first position, thecavity 10 is in communication with a bore 90 for entrance of the bloodsample 8 into the cavity 10 by capillary forces, and, in a secondposition, the cavity 10 is in communication with the liquid storagechamber 1 and a mixing chamber 3 for discharge of the blood sample 8diluted by the liquid diluent 11 into the mixing chamber 3. The mixingchamber 3 is separated by a membrane according to the present inventionwith an orifice 59 from and a collection chamber 5 for the passage ofthe blood sample 8 between the mixing chamber 3 and the collectionchamber 5. The membrane containing the orifice 59 constitutes a part ofa flow-through sensor 4.

A volume metering arrangement is connected to the collection chambercomprising a volume metering chamber 6 having the size of the volume tobe measured during the measurement with two connecting channels 12, 13of relatively diminutive internal volumes for registering liquid entryand exit by optical or electrical means, from the volume meteringchamber a channel 7 leads out to a connection port 67 where a pressurecan be applied.

FIG. 2 schematically illustrates counting and sizing of particles byimpedance determinations. FIG. 2 shows a cross-section of a part of themembrane 91 containing the orifice 59. Two chambers, the mixing chamber3 and the collection chamber 5 communicate through the orifice 59. Thechambers 3, 5 contain electrodes 61, 62 for generation of an electricalfield between them. The membrane 91 is an electrical isolator and thus,the orifice 59 restricts the electrical field whereby a sensing zone 60is established through which particles 58 are aspirated. In the sensingzone 60, each particle 58 will give rise to a displacement of thesurrounding electrolyte, thus blocking part of the current-path betweenthe electrodes 61, 62 thereby generating a short voltage pulse 57. Byaspiration of particles, preferably one by one, through the orifice 59,the particles 58 can be characterized with respect to volume andconductivity by registration of the respective voltage pulse 57characteristics.

FIG. 3 schematically illustrates an apparatus with the disposablecartridge, a docking station and a reader. The chambers on each side ofthe flow through sensor have electrodes 34, 35 extending from anexternal terminal 61, 62 through the base wall 63 of the disposable unitand into a configuration facing the inside of its respective chamber.The cartridge is placed in a docking station 66 in a portable apparatusin order to carry out the test. The docking station 66 has a cup shapedhousing having a base 70 and a circumambient sidewall 71. In the base 70there are respective spring loaded electrical connectors 64, 65 forcontacting the terminals 61, 62 of the cartridge automatically when thecartridge is received as a push fit into the docking station. There isalso a conduit 68 passing through the base wall 70 aligned with theconduit 67 of the cartridge. Conduit 67 at its opening into the upperface of the wall 70 has a seal 69, such as e.g. and O-ring for forming agas tight connection with the lower face of the base wall 63 of thecartridge. A vacuum pump 72 is connected by a line 73 to the lower endof the conduit 68. In a modification of the apparatus, the vacuum pump72 can be reversed so as to apply positive gas pressure to the conduit68.

Schematically indicated at 74 are the further conventional components ofa Coulter counter including all the electronic circuitry and displayequipment needed for the operation of the apparatus.

FIG. 4 Example 1 Sizing of Polymer Beads

A mixture of 5 μm and 10 μm particles suspended in electrolyte wasaspirated through the orifice of the apparatus shown in FIG. 3. Thenumbers of particles detected and the size of each detected particlewere recorded. A bimodal distribution of detected particle size isclearly seen in the figure.

FIG. 5 Example 2 Red Blood Cell Counting

Measurement of blood cells has been performed and the result is shown inFIG. 5. Red blood cells are normally around 5 to 7 μm in diameter andare the most frequent in whole blood, as can be seen on the FIG. 5. Thedistribution is a Gaussian curve, as it should be expected. Blood countscan be used in clinical diagnostics. It is fairly simple to counterythrocytes, leukocytes and thrombocytes by impedance measurements,which are considered the basic parameters for haematology (see“Fundamentals of Clinical Haematology”, Stevens, W.B. Saunders Company,ISBN 0-7216-4177-6).

FIG. 6 Example 3 White Cell Counting Using a Diluent Containing aReagent-Composition Selected so as to Preserve All Blood Cells

Material

Cartridge and apparatus containing the functions as described in thepresent invention,

Isoton, Beckman Coulter (prod. no. 24655) containing: sodium chloride7.9 g/L, potassium chloride 0.4 g/L, disodiumhydrogenphosphate 1.9 g/l,sodiumdihydrogenphosphate 0.2 g/L, disodium-EDTA 0.4 g/L and sodiumfluoride 0.3 g/L.

Vacutainer, K3E, Becton & Dickinson, prod. No. 367652.

Bayer, ADVIA-120 equipment.

Performance

The full sequence of the procedure was as follows:

-   -   Collection of a venous blood sample in a vacutainer tube.    -   Leaving the sample, for the sedimentation process to proceed,        for three hours.    -   Extraction the plasma phase with the major part of the        buffy-coat section included    -   Performing analysis using the Bayer Advia 120 equipment for        obtaining a comparative value for the content of leukocytes.    -   Adding 5.00 ml isotonic solution as diluent to the chamber of        the test rig    -   Adding 10.0 μl of sample to the chamber    -   Mixing liquids in the chamber    -   Starting test sequence on the computer (starts the pump and        readies the sampling)    -   When the liquid reaches the first level electrode sampling is        started    -   When the liquid reaches the second level electrode the sampling        is stopped    -   Sampled values are saved in a file    -   The file is opened with a “pulse-viewer” for data analysing and        calculation of the result using a method of calculation        involving subtraction of, with the leukocytes overlapping red        blood cells.        Results

Bayer Advia-120: 11.96×10^9 leukocytes/L

Test-rig: 11.92×10^9 leukocytes/L

Difference in accuracy: (11.96−1.92)/11.96=0.33%

FIG. 7 Example 4 White Cell Isolation Using a Diluent Containing aReagent Composition Selected so as to Primarily Hemolyse the Red BloodCells

Material

Cartridge and apparatus containing the functions as described in thepresent invention,

Diluent containing: procaine hydrochloride 0.10 g/L, 1,3-dimethylolurea0.90 g/L, N-(1-acetamido)iminodiacetic acid 1.28 g/L, dodecyltrimethylammonium chloride 7.51 g/L and sodium chloride 0.03 g/L.

Vacutainer, K3EDTA, Becton & Dickinson, prod. No. 367652.

Performance

The full sequence of the procedure was as follows:

-   -   Collection of a venous blood sample in a vacutainer tube.    -   Leaving the sample, for the sedimentation process to proceed,        for three hours.    -   Extraction the plasma phase with the major part of the        buffy-coat section included    -   Adding 2,000 ml diluent as described above to the chamber of the        test rig    -   Adding 4.0 μl of sample to the chamber    -   Mixing liquids in the chamber    -   Starting test sequence on the computer (starts the pump and        readies the sampling)    -   When the liquid reaches the first level electrode sampling is        started    -   When the liquid reaches the second level electrode the sampling        is stopped    -   Sampled values are saved in a file    -   The file is opened with a “pulse-viewer” for data analysing and        generation of the result.        Results

As can be seen in the histogram in FIG. 6 the particle populationcorresponding to the leukocytes is easily identified in the absence ofthe red blood cells.

FIG. 8 Example 5 Counting Somatic Cells

Milk quality is essential for farmers, diary producers and consumers.Farmer has to deliver milk of a certain quality, which is controlled bythe so-called Somatic Cell Count (SCC). In milk quality tests somaticcells in the milk are counted to determine infections (clinicalmastitis). A limit of 400,000 cells pr. ml. has to be met by the farmersfor dairy resale. Change of diet, stress or mastitis lead to higher SCClevels, thus lowering the quality of the milk and consequently loweringthe price per unit volume. A cheap cell counter will help farmers anddiary producers monitor SCC-level.

As schematically illustrated in FIG. 9, orifices 59 for Impedance cellsizing can be fabricated by laser micro machining of polymers 91 leadingto a simple and convenient way of fabricating and assembling orifices 59for the cartridge. A series of small orifices of 50 μm has beenfabricated with an UV-laser 100. The orifices 59 are made in less than10 ms in a 50 μm polymer sheet. The uniformity of the orifices 59 isvery high and the smoothness of the orifice entrance is unique.

Preferably, the laser 100 is a UV-laser, such as an excimer laser with awavelength in the range from 150 nm to 350 nm, because of its superiorlaser cutting accuracy.

Manufacturing of polymer membranes 91 with orifices 59 by laser cuttingaccording to the present invention provides cheap and rapid productionof membranes 91 with precision-machined orifices 59 for impedanceparticle counting and/or sizing.

The high-energy laser spot 104 causes vaporization or ablation of thematerial in the focused region of the spot 104. The laser spot 104 of anexcimer laser 100 may be focused to a few micrometers, providing thedesired accuracy in accordance with the present invention.

In the embodiment illustrated in FIG. 9, the laser 100 is used like aconventional drill, i.e. the focussed laser spot 104 remains at thedesired position of the orifice and the orifice 59 is produced by aseries of laser pulses.

In the embodiment illustrated in FIG. 10, the focussed laser beam 102 isscanned along the desired circumference of the orifice 59 therebycutting-out the orifice 59 of the membrane 91. In this way any desiredcircumferential shape of the orifice 59 may be manufactured.

According to yet another embodiment of the invention, a narrow laserbeam 102 is scanned for example linearly, e.g. line by line, across thesurface of the membrane 91 desired to be removed for creation of theorifice 59.

FIG. 11 shows a cross-section of a membrane orifice 59 manufactured inaccordance with the present invention. The illustrated polymer membrane91 is provided with an orifice 59 with rounded edges 56 at one of thesides of the membrane 91 whereby perturbations of an electrical field atthe orifice 59 entrance are minimised and a substantially homogenouselectrical field at the centre of the orifice 59 may be provided.

Hereby, electrical pulses generated by particles passing the orifice 59at the centre of the orifice 59 and particles passing the orifice 59close to an edge of the orifice 59 will generate substantially identicalpulses. Without rounded edges 56, particles passing the orifice 59 closeto an edge will generate a distorted pulse.

Preferably, the radius of curvature of the rounded edges 56 correspondsto ¼th the diameter of the orifice 59.

1. An electrical impedance cell counting apparatus for counting andcharacterizing particles suspended in a liquid, comprising: a housingwith a mixing chamber and a collection chamber separated by a polymermembrane containing an orifice for passage of the particles between themixing chamber and the collection chamber, wherein a diameter of theorifice is in a range from 10 μm to 1,000 μm; electrodes within themixing and collection chambers configured to provide pulses by impedancedetermination for counting the particles that pass through the orifice;and a volume meter that determines a period during which a fixed volumeof the liquid passes through the orifice.
 2. An electrical impedancecell counting apparatus according to claim 1, wherein the orifice hasrounded edges at one of the sides of the membrane whereby perturbationsof an electrical field at an orifice entrance are minimized and asubstantially homogenous electrical field at the center of the orificeis provided.
 3. An electrical impedance cell counting apparatusaccording to claim 2, wherein a radius of curvature of the rounded edgesis substantially equal to ¼^(th) the diameter of the orifice.
 4. Anelectrical impedance cell counting apparatus according to claim 1,wherein a surface roughness of an internal surface of the orifice is ina range from 0 μm to 5 μm, whereby a substantially homogenous electricalfield at a center of the orifice is provided.
 5. An electrical impedancecell counting apparatus according to claim 1, wherein the orificediameter is in a range from 30 μm to 75 μm.
 6. An electrical impedancecell counting apparatus according to claim 1, wherein the orificediameter is 50 μm.
 7. An electrical impedance cell counting apparatusaccording to claim 1, wherein a length of the orifice ranges from 1 μmto 1000 μm.
 8. An electrical impedance cell counting apparatus accordingto claim 1, wherein the membrane is positioned in a single-usecartridge.
 9. An electrical impedance cell counting apparatus accordingto claim 1, further comprising: a bore in an outer surface of thehousing for entrance of a liquid sample, communicating with a samplingmember positioned in the housing for sampling the liquid sample andhaving a cavity for receiving and holding the liquid sample, thesampling member being movably positioned in relation to the housing insuch a way that in a first position the cavity is in communication withthe bore for entrance of the liquid sample into the cavity, and in asecond position the cavity is in communication with the mixing chamberfor discharge of the liquid sample into the mixing chamber.
 10. Anelectrical impedance cell counting apparatus according to claim 1,wherein deviation of the orifice diameter along a longitudinal axis ofthe orifice ranges from +/−1% to +/−10%, whereby a substantiallyhomogenous electrical field is provided at a center of the orifice. 11.An electrical impedance cell counting apparatus according to claim 1,wherein a largest cross-sectional dimension of the orifice is from 10 μmto 50 μm.
 12. An electrical impedance cell counting apparatus accordingto claim 1, wherein the volume meter comprises: a volume meteringchamber having an entrance connected to the collection chamber, andhaving a detector that detects liquid at the entrance and at an exit ofthe volume metering chamber.
 13. An electrical impedance cell countingapparatus according to claim 12, wherein the detector comprises anoptical detector.
 14. An electrical impedance cell counting apparatusaccording to claim 12, wherein the detector comprises an electricaldetector.
 15. An electrical impedance cell counting apparatus forcounting and characterizing particles suspended in a liquid, comprising:a housing with a mixing chamber and a collection chamber separated by apolymer membrane containing an orifice for passage of the particlesbetween the mixing chamber and the collection chamber; and electrodeswithin the mixing and collection chambers configured to provide pulsesby impedance determination for counting the particles that pass throughthe orifice, wherein a diameter of the orifice is in a range from 10 μmto 1,000 μm, and wherein a diameter of the particles is not greater than60 percent of the diameter of the orifice.
 16. An electrical impedancecell counting apparatus according to claim 15, wherein the orifice hasrounded edges at one of the sides of the membrane whereby perturbationsof an electrical field at an orifice entrance are minimized and asubstantially homogenous electrical field at the center of the orificeis provided.
 17. An electrical impedance cell counting apparatusaccording to claim 16, wherein a radius of curvature of the roundededges is substantially equal to ¼^(th) the diameter of the orifice. 18.An electrical impedance cell counting apparatus according to claim 15,wherein a surface roughness of an internal surface of the orifice is ina range from 0 μm to 5 μm, whereby a substantially homogenous electricalfield at a center of the orifice is provided.
 19. An electricalimpedance cell counting apparatus according to claim 15, wherein themembrane is positioned in a single-use cartridge.
 20. An electricalimpedance cell counting apparatus according to claim 15, furthercomprising: a bore in an outer surface of the housing for entrance of aliquid sample, communicating with a sampling member positioned in thehousing for sampling the liquid sample and having a cavity for receivingand holding the liquid sample, the sampling member being movablypositioned in relation to the housing in such a way that in a firstposition the cavity is in communication with the bore for entrance ofthe liquid sample into the cavity, and in a second position the cavityis in communication with the mixing chamber for discharge of the liquidsample into the mixing chamber.
 21. An electrical impedance cellcounting apparatus of claim 15, wherein the diameter of the particles isbetween 5 to 25 percent of the diameter of the orifice.
 22. Anelectrical impedance cell counting apparatus of claim 15, furthercomprising a volume meter that determines a period during which a fixedvolume of the liquid passes through the orifice.
 23. An electricalimpedance cell counting apparatus according to claim 15, wherein thevolume meter comprises: a volume metering chamber having an entranceconnected to the collection chamber, and having a detector that detectsliquid at the entrance and at an exit of the volume metering chamber.24. An electrical impedance cell counting apparatus according to claim23, wherein the detector comprises an optical detector.
 25. Anelectrical impedance cell counting apparatus according to claim 23,wherein the detector comprises an electrical detector.
 26. A single-usedisposable electrical impedance cell counting cartridge for counting andcharacterizing particles suspended in a liquid, comprising a housingwith a mixing chamber and a collection chamber separated by a polymermembrane containing an orifice for passage of the particles between themixing chamber and the collection chamber for impedance determination ofthe particles, wherein a diameter of the orifice is in a range from 10μm to 1,000 μm.
 27. A single-use disposable electrical impedance cellcounting cartridge of claim 26, further comprising a volume meter fordetermining a beginning and an end of a period during which a fixedvolume of the liquid passes through the orifice.
 28. A single-usedisposable electrical impedance cell counting cartridge of claim 27,wherein the volume meter comprises: a volume metering chamber having anentrance connected to the collection chamber, and having an exit,wherein the entrance and the exit of the volume metering chamber areconfigured for detecting presence of the liquid at the entrance and atthe exit.
 29. A single-use disposable electrical impedance cell countingcartridge of claim 27, further comprising electrodes within the mixingand collection chambers configured to provide pulses by impedancedetermination for counting the particles that pass through the orifice.